Gene amplification is one of the major mechanisms of altered gene expression in human cancers. However, the targets of gene amplification are only partially characterized. We have developed a novel approach to the identification and characterization of amplified regions based on chromosome microdissection. This technology has been applied to the chromosomal abnormalities (homogeneously staining regions and double minutes) associate with gene amplification in tumor cells from breast, ovarian and prostate carcinomas. Using probes generated by microdissection for fluorescence in situ hybridization, we have identified a series of chromosomal regions involved in amplification events not previously recognized in these diseases. In addition, it has been possible to determine the complex structure of homogeneously staining regions in several cases establishing that these structures are frequently composed of DNA segments derived from multiple chromosomes and intermingled to form an abnormal region. In order to isolate candidate target genes from these regions, we have developed a technique based on hybrid selection of CDNAs using the microdissected material This technology, which supplements standard positional cloning techniques, has been utilized to isolate candidate genes from amplified regions on chromosome 17 and 20 in breast cancer, as well as chromosome 12 in sarcomas. In addition, it has been possible to apply these extremely robust microdissection probes to evaluate clinical material for the presence of gene amplification by FISH in prostate cancer and sarcomas. The methodology previously described illustrates the importance of developing rapid techniques for the identification of genes amplified in a series of key human tumors. A specific example of the importance of this work relates to our recent studies of the cytologically recognizable sites of gene amplification in human breast cancers. This study illustrated the utility of microdissection to identify both the composition and origin of chromosomal material amplified within chromosome rearrangements in this important malignancy. In addition to recognizing known sites of gene amplification, the study identified several previously unidentified regions amplified in breast cancers. This finding will allow the utilization of various cDNA hybrid-selection, and direct hybridization approaches to identify genes over-expressed due to a copy number change. Because of the clinical significance of gene amplification in various malignancies (including breast and prostate cancer), it appears highly likely that important new information will be discovered relative to genes playing a causal role in disease genesis or progression.